JPS60155121A - Therapy for acquired immunological dysfunction syndrome - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for treating acquired immunodeficiency syndrome.

(Prior art) Primary immunodeficiency is extremely rare, but
It is a fatal condition in which part or all of the body collapses or one or more immune responses are completely absent. It is classified as a life-ending, recurrent, chronic infectious disease that requires the victim to survive under strictly controlled, sterile conditions.

Therefore, there is no treatment other than isolating the patient from all potential sources of infection.

Secondary immunodeficiency is manifested as the function of the immune system deteriorates due to factors such as aging, radiation or chemotherapy for cancer cells. Patients with such immunodeficiency (insufficient or incomplete immune function) are easily infected by viruses and other pathogens, and have lost their immune system's resistance, and countermeasures for this are difficult and long-term. It is.

Acquired immunodeficiency syndrome (AIDS) (so-called AIDS)
is a recently identified syndrome in which the normally functioning human immune system loses its proper function. Immunodeficiency syndromes are somewhat similar to the clinical pathology of secondary immunodeficiency syndromes, and are characterized by a reduced rate of helpers that suppress a subpopulation of cells.

The etiology of acquired immunodeficiency syndrome is unknown, and no effective treatment has been discovered.

Due to the destruction of the immune system, patients with acquired immunodeficiency syndrome are susceptible to disease, resulting in high infection rates and high mortality rates from Kaposi's sarcoma. References include “Immunocompromised Homorefcial” Lancet 19
81; ii: 1325-6; Center for Disease Control, “Epidemiological Aspects of the Current Outbreak of Capacitive Sarcoma and Opportunistic Infection” 1982.306:248 -52; Severely Acquired Immunodeficiency in European Homosexual Men'' 1 by Gerstoft et al.
982.285:17-19, etc. The two-year mortality rate is reported to be as high as 80%.

What is known so far is that the thymus gland is connected to the immune system of the body, and there has been a great deal of interest in substances isolated from the thymus gland. Certain lymphocytes divide in the thymus, leaving behind cells from the thymus called T-cells, which circulate in the blood and reach the lymph, tiles, and lymph nodes. References include U.S. Patent No. 4,002,602
;4,010,148;4.082,737.4,14
8,886.4, 167.557, etc.

Attempts to restore cell-mediated immunity by reviving thymus-dependent T-cell function have used a variety of thymic extracts of various chemical, physical, and biological organisms. This has been done through animal and living experiments. References include "Pathymic Hormone: Biochemistry and Biological and Clinical Activity" 17, 281 (1977);
“The Role of Thymic Hormones in Regulation of the Lymphatic System” 83-102 (1
977). It has been observed that 6-degree purified thymic factor will not be able to elicit a cell-dependent immune response in immunodeficient animals either inside or outside the organism. References include ``Immunodeficiency in Human Diseases in Nature and Ethological Significance,'' presented at the Tokyo Symposium on September 13-15, 1976. and support the theory that several protein hormones contained in thymus extracts are necessary for a moderate range of survival activities.

It has been found that certain thymus extracts (hereinafter referred to as "'TP-1") can be effectively used to stimulate the cellular immune system of patients with acquired immunodeficiency syndrome. This discovery is surprising because the etiology and prevention methods for acquired immunodeficiency syndrome are unknown.

Therefore, an object of the present invention is to provide a method for stimulating and activating the cellular immune system in patients with acquired immunodeficiency syndrome.

(Summary of the Invention) This invention provides for the treatment of acquired immunodeficiency syndrome, particularly for humans with acquired immunodeficiency syndrome.
The present invention relates to a method for activating a cellular immune system by administering an immune-enhancing effective amount of TP-1. And when this extract is electrophoresed using polyacrylamide gel at pH 8.6, it has an Rf of about 0.25 and 0.44.
Two main characteristic band spectra with .

(Description of the invention) TP-1 is a calf thymus hormone extract.
It is described in the paper "Isolation, behavior and biological effects of calf thymus factor" at the 3rd European Society of Immunology held in Copenhagen, August 25-21, 1976. Other references include ``Calf thymus extract (
"Pharmaceutical and biological properties of TP-1)" 1977, 3
(2) 39-47: “Chemical behavior and biological activity of a new thymus extract” and others. Also, TP-
1 is manufactured by Serono Laboratories, Inc.

Extraction and purification of TP-1 was carried out using the above-mentioned ``Calf thymus extract''.
According to this article, thoroughly homogenized calf breast 11I (thymus body) is extracted with 0.158 ammonium acetate. The collected liquid was heated at 70°C for 3'0 minutes.

Proteins coagulated by this method were separated by filtration and precipitated by addition of nanmonium sulfate until a clear liquid reached 45% saturation. The clear liquid obtained by centrifugation was saturated to 90% with ammonium sulfate. This final isolate is collected by filtration, dissolved in water, and PM-10
Uru + filtration was performed using a membrane.

The ultra filtrate has a molecular weight of about 10. Contains protein substances below Goo, which are 1. It was made lyophilic, desalted on Sephadex G-25, and gel-filtered on Sephadex G-50. The small fraction collected was approximately 0.25 to 0 when electrophoresed using a pH 8.6 polyacrylamide gel compared to promogenol blue used as a tracer.
．． It showed two characteristic band spectra with an Rf of 44.

TP-1 stimulates phytohemagglutinin (PHA) and concanavalin type A (CO1'1CanaValinAS).
It has been found to have the ability to enhance the reactivity of mouse tile lymphoid cells to stimulation (stimulation).

Ru. On the other hand, ribopolysactucharide stimulation (ttpopo+y
-5accharideStimulation) does not enhance the response. Also, the human code (cord,...
Stimulating the addition of E- rosette-forming lymphocytes from the tissue (blood) and increasing the percentage of Φ-theta-positive cells in the mouse tile population (AIIOilleneiC).
1! > stimulates bone marrow cell capacity in mice with X
Ray radiation is what triggers the graph-to-host response in mice. ,,,.

TP-1 had no acute toxic effect and was administered to the internal peritoneum of mice for 21 days and to the internal peritoneum of rats for 31 days.
There are no side effects even when administering up to 100197kQ, furthermore, there are no side effects even when administering up to 1g/kg of 50II subcutaneously to rats for 180 days, and 101
! There were no side effects even when administered up to II/kll. In addition, TP-1 did not alter nerve and muscle conduction assessed in vitro on the diaphragmatic nerve in rats or in vivo on the tibial and sakaki meatus in mice; blood pressure, electrocardiography, and respiration. It does not change the motor pattern.

TP-1 can be easily distinguished from other thymus extracts. It is found that when TP-1 is electrophoresed using a polyacrylamide gel at DH8,6, approximately 0.2
This is because two main characteristic band spectra appear with Rf of 5 and 0.44, whereas other thymus extracts show only a single characteristic band (characteristic band spectra). It is. At pH 8.6, TP-
The two band spectra (bands) of 1 are Rf=0125
±0.05 and Rr=0.45±0.05.

To date, TP-1 has been administered by intramuscular injection, but other methods can also be used. TP-1 with a compatible carrier or diluent; for intramuscular injection, sterile water.
A medicament can be formulated containing an immunostimulating effective amount of. In addition, supplementary drugs such as mannitol may be used, and other dosage forms may also be used. Preferably, the composition for intramuscular injection contains about 5 to 25 mg of protein per portion.

The effective immune-stimulating amount of TP-1 is determined by the age and weight of the patient, the method of administration, and the presence or absence of Kaposi's sarcoma or other disease infection; the daily dose is approximately 0.
．． A range of 5 to about 1.0 mo/kcl is typical, but responses vary considerably from individual to individual and are best determined by the clinician.

TP-1 was studied in vitro in blood samples of various patients diagnosed with acquired immunodeficiency syndrome and in control patients. Initial studies were performed on phytohemagglutinin (plant lectin) (PHA) stimulation. This method is based on the fact that peripheral blood lymphocytes, present as monocytes, divide upon stimulation of PHA, and the dividing cells are thymic lymphocytes. The degree of division can be measured by introducing 3H-thymidine into the ribonucleic acid (DNA) of the cell.

Calculate the lymphocytes contained in serum-free Hank's Balanced Salt Solution (+-IBSS),
For the T-cell rosette test, after removing the cells, the remaining cells were grown at 2x10/2 in additional medium (RPHI-1640 with 251H Heves buffer).
Resuspend at the if concentration. Cell aliquots are PHAMffi per lymphocyte (0.5 g,

2uQ, or 10uo,)10u1. added to. 4 x 1051 DI11 per aliquot were added in 5 doses, incubated for 72 hours at 37°C in a 5% CO2 humidified atmosphere, labeled with 0.5uCi3H-methyl-thymidine, and then incubated for 4.5 hours. moreover,
Cells are harvested and spread onto grids in amounts of 10-411.

The average stimulation value recorded is the PHA triple dose height. The results are shown in the table below.

In a controlled study, two people showed a negative rate of increase and four people showed a positive rate of increase (up to 16.20.7 and 36x, respectively) on all doses of TP-1, but each person
There was a negative increase in the dose of 1250 icg.

A second study was conducted on opposite sex factor stimulation. The basis of this research is based on the following facts. That is, when stimulated by cells of the opposite sex, dividing mononuclear cells (
In the presence of monocyte divides, peripheral blood lymphocytes and their division rate are absorbed into DNA by radioactive a
This fact can be determined by quantifying thymidine. After counting the lymphocytes in serum-free Hank's Balanced Salt Solution (HBSS) and removing those cells for the ■-cell rosette test, the remaining cells were added to additional medium (25 ■H Resuspend in RPHI-1640 containing Heves buffer at a concentration of 2xlO6/ugly1. Aliquots of control cells were irradiated with 3.000 rads, and for each medium 2xlO5-reactive cells (
0.1m1. Medium) are mixed with sterilized 2X105 sting-irradiated cells (0,11, medium) over a period of five minutes. The medium is
Irradiated for approximately 120 hours at 37°C in a 5XC02 humidified atmosphere and 0.5u in 50u1 of phosphate buffer.
Labeled with Ci3H-medyruthymidine, then 1
Re-incubate for 8-24 hours. Cells are then collected and counted.

1975 New England Journal of
See Medicine, 292.70. The results are shown in the table below.

The results of a controlled study on serum from five people without acquired immunodeficiency syndrome are shown in the table below.

,... ,,・Control patient.

TP-1 was used in vivo in a 43-year-old man with acquired immunodeficiency syndrome. The man had his first symptoms in January 1981, complaining of a rash, discomfort, alopecia, and weight loss over a one-year period. The diagnosis revealed symptoms of ichthyosis, alopecia, and lymphadenopathy. After that, medical examinations were carried out for -2 years, but no firm diagnosis was made until the end of 1981, when an abnormal 0KT4:0
Lymphocyte marker studies showed symptoms of T-cell lymphopenia in KT8. 0KT4.0KT8 is a monoclonal antibody specific for ■-cell subpopulations, namely the human inducible/helper knee lymphocyte subclass and the human suppressive/cytotoxic T-lymph cell subclass. Said concentrate combined with abnormal immunoglobulin [I
gA7.60/+ (normal range 0.5-3g/l) ICI
H3,3o/l (normal range 0.5-1.7g/l) and I(IG8.3 (1/l (normal range 5-16g/l)
J and liver function test [731U/+ (normal range 17~
The diagnosis is based on the asparatase aminotransferase activity, which was regularly increased to 351 U/l), and the results of clinical photographs.

In March 1982, a patient presented with symptoms of fever, stiffness, difficulty breathing, and cough. Chest radiography showed a widely diffuse infiltrate. The patient refused bronchoscopy and was subsequently diagnosed with pneumonia caused by a protozoan parasite that lives in the lungs of domestic rabbits. Parental cotrimoxazole was started (trimethoprine 2011 g/kg and sulfamethoxazole 100 ma/ko) and the patient improved. 100
The patient's lymphocytes mixed with TP-1 in mg/I concentrate were cultured overnight in vitro to obtain a normal proportion of OKT4+ cells, thereby providing the basis for using TP-1 in vivo. Therefore, immediately after the above-mentioned cotrimoxazole, Ding P-1
o, 75 mo/kq was used intramuscularly every other day.

The giant cell virus was searched for and was repeatedly isolated from gargled fluid and urine.

On March 11th, the number of cells was 15X10'/l, on April 16th, the number of cells increased to 100x10'/l, and on April 23rd, the number of cells was 121X104.
/l increased. Between April 23 and May 5, 1982, the patient was diagnosed with candidiasis. TI)-1
treatment was stopped after a few days, at which time the lymphocyte counts had fallen to pretreatment values.

In June of that year, the patient died from encephalitis that was resistant to chemotherapy, including antivirals. A lack of immune stimulation that supports lymphocytes is suspected to be the cause of his severe illness.

Continued from page 1 [Phase] Inventor Charlotte Nis Redden o Inventor Aliza Eshkol 0 Inventor Gerald E. Stiles 18 Glenwood Street, Natick, Massachusetts, United States of America 17 Emekli Frame, Tel Hashomer, Israel, United States of America 66 Pryor Farm Road, Goxbury, Massachusetts

Claims (1)

[Scope of Claims] (1) When a person with acquired immunodeficiency syndrome 1 was subjected to electrophoresis using a polyacrylamide gel at pH 8.6, two molecules with Rf of approximately 0.25 and 0.44 were detected. It is characterized by the activation of the immune system by administering an effective dose of TP-1, a calf thymus hormone extract, in which two main characteristic band spectra appear. How to treat acquired immunodeficiency syndrome. (2) The method for treating acquired immunodeficiency syndrome according to claim 1, wherein the calf thymus hormone extract is administered parenterally. (3) The method for treating acquired immune deficiency syndrome according to claim 2, wherein the calf thymus hormone extract is administered intramuscularly.・,′ (4) Calf thymus hormone extract administered intramuscularly is
4. A method of treating acquired immunodeficiency syndrome according to claim 1, wherein the protein remains at a vk weight of from about 5 to about 251 o/ml.゛(5) Gamma cow thymus-hormone extract'--daily amount of
The method for treating acquired sexual immunodeficiency syndrome as described in claim 4, wherein the dose is 1&j'', about 0.5 to about 1.0110/kg. ``'''' (6)+゛The acquired immunodeficiency according to claim 3, wherein the bovine thymus phophremone extract is in one concentration containing 5 proteinaceous proteins. How to treat the syndrome.゛...,. (7) The daily dosage of calf d thymus hormone extract is from about 6.5 to about 1.000 sa/kg. : arrow i″ immunodeficiency]′
The treatment method for all syndromes: the two-day administration of Repar-■...''..., (g') Fertility Extract, Polmon Extract, is about 6 to about 0. "'1g/'Q' in a certain patent claim... Scope - '1' mound/record [Method for treating acquired immunodeficiency syndrome. (9) Polyacrylamide gel (mouth → In IH potato, 1 and 6, Boria'acrylamide gel No. ``Koji:Ide:'' when electrophoresed, J was about 0.25 and 0.
．． A calf thymus hormone extract that activates the cellular immune system, and contains two main characteristic bands: 44'R's and R's.